A Review of Sperm Ultrastructural Characters in the Opecoelidae (Digenea) and Their Phylogenetic Implications, with New Data on Peracreadium characis, a Parasite of Diplodus puntazzo in Tunisia

Simple Summary The mature spermatozoon of Peracreadium characis, an intestinal parasite of the sheephead bream, is described by means of transmission electron microscopy (TEM). It shares the common ultrastructural features described in the majority of digeneans (two 9+‘1’ axonemes, mitochondria, nucleus and parallel cortical microtubules). The absence of external ornamentation of the plasma membrane and spine-like bodies distinguishes the spermatozoon of P. characis from the spermatozoa of the remaining studied opecoelids. Abstract The spermatozoon ultrastructure of Peracreadium characis (Stossich, 1886) (Digenea: Opecoelidae), an intestinal parasite of the sheephead bream Diplodus puntazzo (Walbaum, 1792) (Sparidae), is described by means of transmission electron microscopy (TEM). The mature spermatozoon possesses two axonemes of the 9+‘1’ trepaxonematan pattern, an anterior electron-dense material, two mitochondria, a nucleus and parallel cortical microtubules distributed in two bundles. The absence of external ornamentation of the plasma membrane and spine-like bodies are the noteworthy characters that distinguish the spermatozoon of P. characis from those of most opecoelids. In fact, only Helicometra fasciata lacks external ornamentation in the spermatozoon. A comparative study with the remaining opecoelids described so far reveals similarities in the ultrastructural organization of their sperm cells. In addition, the current data on sperm ultrastructure in species of the recognized opecoelid subfamilies are compared, namely the Hamacreadiinae, Helicometrinae, Opecoelinae, Opistholebetinae and Plagioporinae.


Introduction
The cosmopolitan digenean family Opecoelidae Ozaki, 1925 is a species-rich family with more than 90 genera and almost 900 species, parasitizing the digestive tract of marine and freshwater teleost fishes and also sporadically found in amphibians [1].
This study presents the first description of the sperm cell of the opistholebetine Peracreadium characis (Stossich, 1886), thus increasing the information on sperm ultrastructure in the Opecoelidae. Furthermore, some criteria potentially useful for phylogenetic analysis are discussed by comparing the obtained results with data from other Digenea, particularly other opecoelidean species.
This study presents the first description of the sperm cell of the opistholebetine Per acreadium characis (Stossich, 1886), thus increasing the information on sperm ultrastructur in the Opecoelidae. Furthermore, some criteria potentially useful for phylogenetic analy sis are discussed by comparing the obtained results with data from other Digenea, partic ularly other opecoelidean species.

Specimens
Fifty-two sheephead breams Diplodus puntazzo (Walbaum, 1792), captured in th Mediterranean Sea off La Chebba (34°14′ N, 11°06′ E) (Tunisia) in September 2016, Marc 2017 and May 2018, were scanned for helminths in their digestive system. Live adult d geneans were isolated from the intestinal tract, and some of them were stained with Sem ichon's acetic carmine and mounted in Canada balsam (Figure 1a,b). The mounted spec mens were then identified as P. characis in agreement with the specialized literature [2,39 Two slides with three specimens of P. characis ex. D. puntazzo from La Chebba (Tun sia) were deposited in the Muséum National d'Histoire Naturelle (Paris, France) unde the accession numbers MNHN HEL1930 and MNHN HEL1931.

Transmission Electron Microscopy
Living adult digeneans were rinsed immediately upon removal from the fish with a 0.9% NaCl solution. They were then fixed in 2.5% glutaraldehyde at 4 • C in a 0.1 M sodium cacodylate buffer (pH 7.4) for a period no shorter than 2 h. After rinsing in a 0.1 M sodium cacodylate buffer (pH 7.4), the digeneans were postfixed in 1% osmium tetroxide at 4 • C with 0.9% potassium ferricyanide in the same buffer for 1 h. After rinsing in Milli-Q water (Millipore Gradient A10, Millipore Co., Merck KGaA, Darmstadt, Germany), the dehydration process began using an ethanol series and propylene oxide. The specimens were finally embedded in Spurr's epoxy resin and polymerized at 60 • C for 72 h. Ultrathin sections 60 nm thick were obtained using a Leica Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany), placed on copper grids and double-stained with uranyl acetate and lead citrate, as in Reynolds [40]. Stained grids were observed under a JEOL 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan), operated at an accelerating voltage of 80 kV, in the Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB).

Cytochemistry
The Thiéry [41] technique was applied to allow for the cytochemical detection of glycogen. Ultrathin sections in gold grids were treated in periodic acid, thiocarbohydrazide and silver proteinate (PA-TCH-SP) in the following sequence: 30 min in 10% PA, rinsed in Milli-Q water, 24 h in TCH, rinsed in acetic solutions and Milli-Q water, 30 min in 1% SP in the dark and rinsed in Milli-Q water. Sections were examined with a JEOL 1010 transmission electron microscope (JEOL Ltd.) in the CCiTUB.

Results
The ultrathin sections of P. characis spermatozoa reveal three distinct regions (I, II and III) from their proximal to distal extremities, each with distinct ultrastructural characters (Figures 1-3). The cell is filament-shaped, with structures found in many digeneans, including two axonemes, mitochondria, two bundles of parallel cortical microtubules, cytoplasm with glycogen granules and a nucleus.

Region I: Anterior Spermatozoon Extremity (Figures 2a-h and 4I)
The region near the anterior tip of the sperm cell displays one 9+'1' trepaxonematan axoneme as well as an electron-dense material, which disappears when the second axoneme's singlets and doublets appear (  (Figure 3g). The disappearance of the first mitochondrion marks the transition to region II.

Region II: Middle Spermatozoon Region (Figures 2i-k and 4II)
The middle region or region II has two axonemes, parallel cortical microtubules arranged into two bundles and a large amount of glycogen granules in its anterior part (Figures 2i,j and 4II). Another moniliform mitochondrion can be observed in the posterior portion of region II (Figures 2k and 4II). It is noteworthy that the number of cortical microtubules increases from 8 (7 + 5) (Figure 2i) to 15 (7 + 8) (Figure 2k).

Region III: Posterior Spermatozoon Extremity (Figures 2l, 3a-f and 4III)
The proximal part of region III starts with the appearance of the nucleus, which is accompanied by the simultaneous presence of the second mitochondrion and the two axonemes, glycogen granules and cortical microtubules (Figures 2l, 3a and 4III). The character sequence transition toward the posterior tip of the spermatozoon varies among sperm cells. In fact, the second mitochondrion disappears at different levels, either before or after the first axoneme becomes disorganized. As represented in Figure 4III, one type of posterior extremity is characterized by the following sequence of character disappearance: second mitochondrion, first axoneme, second axoneme, nucleus and cortical microtubules ( Figures 3a,b,e,f and 4III). A second type exhibits the following sequence of character disappearance: first axoneme, second axoneme, second mitochondrion, nucleus and cortical microtubules (Figures 3c-f and 4III). The posterior tip of the spermatozoon has only cortical microtubules and granules of glycogen.

Discussion
The mature spermatozoa of P. characis are filiform cells tapered at both ends, which share the common ultrastructural features described in the majority of digeneans: two axonemes, a nucleus, mitochondria and parallel cortical microtubules. However, compared to most opecoelidean species studied, the mature spermatozoon of P. characis presents some unusual features that are briefly discussed below.

Anterior Extremity: Anterior Dense Material
In P. characis, the spermatozoon's anterior extremity displays the first axoneme with a continuous and submembranous layer of an electron-dense material located on the opposite side of the axoneme, which disappears when the two axonemes are completely formed and the cortical microtubules appear. This structural element has been described in all opecoelids studied up to now, except in the opistholebetine Heterolebes maculosus, the opecoeline Opecoeloides furcatus and the plagioporines Nicolla testiobliqua and N. wisniewskii [23,34,37,38].

Axonemes
The axoneme is a significant source of characters for elucidating platyhelminth phylogeny. The shape and length of the axoneme have been the subject of numerous discussions as spermatological characters in Digenea phylogeny. In terms of their morphology, the spermatozoon of P. characis possesses two axonemes exhibiting the 9+'1' trepaxonematan pattern, which is composed of nine peripheral doublets with dynein arms and a highly structured central element [53]. This type of axoneme has been observed in the majority of Digenea sperm cells, with the exception of species of Schistosoma Weinland, 1858 (Schistosomatidae Stiles & Hassall, 1898) and Didymozoon Taschenberg, 1879 (Didymozoidae Monticelli, 1888): the former presents the 9+'1' special pattern (one diffuse central element and nine peripheral doublets with no dynein arms) [54], while the latter presents the 9+0 axoneme pattern (nine doublets with neither outer dynein arms nor central structure) [55].
The axonemes of the male gamete of P. characis present different lengths, as in all opecoelid spermatozoa studied until now. In this species, the disorganization of the first axoneme occurs at the nuclear region of the spermatozoon, similarly to all Opecoelidae species except for Labracetabulum gephyroberici and O. furcatus [33,34], in which the first axoneme disorganizes and disappears at the very anterior part of the nuclear region. Within the Digenea, only the lecithasterid Aponurus laguncula Looss, 1907 and the haplosplanchnid Haplosplanchnus caudatus (Srivastava, 1937) [56,57] show two axonemes with the same length presenting centrioles at the same level in the anterior spermatozoon extremity, and both axonemes also disorganize at the same level in the posterior part of the nuclear region.

External Ornamentation
An external ornamentation of plasma membrane has been reported in the anterior areas of male gametes in many species of the digeneans. This ornamentation does not usually cover the entire perimeter of the cell [26]. The location of the external ornamentation along the spermatozoon varies among species, leading to three groups of digeneans: a group with ornamentation in the apical spermatozoan region; a group with a more posterior external ornamentation, most commonly in the mitochondrial region and another group that includes species presenting spermatozoa without external ornamentation [24]. The spermatozoon of P. characis such as Helicometra fasciata lacks external ornamentation, following Quilichini et al.'s [24] type 3 spermatozoon, in contrast with the remaining Opecoelidae studied to date, which exhibit external ornamentation in the mitochondrial region corresponding to Quilichini et al.'s [24] type 2.

Cortical Microtubules
Different aspects of cortical microtubules are considered crucial in platyhelminth phylogeny inference, including whether they are present or not, how they are aligned, the region where their maximum number is located, the quantity of bundles and if they are associated or not with the external ornamentation of the plasma membrane [26].
The parallel alignment of cortical microtubules has also been found in the sperm cells of most groups of parasitic flatworms, such as the Monogenea van Beneden, 1858 (except in certain monopisthocotyleans) [27,59,60] and the Eucestoda Southwell, 1930, except for the Cyclophyllidea van Beneden in Braun, 1900 and Tetrabothriidea Baer, 1954, which have twisted cortical microtubules [25].
Among the majority of digeneans, the cortical microtubules are typically arranged into two bundles in the sperm cell. This was also observed in P. characis, similarly to other opecoelidean species studied to date. However, some hemiuroidean species (such as the lecithasterids, hemiurids and sclerodistomids) exhibit only one bundle of cortical microtubules on the ventral or mitochondrial side of the sperm cell [56,[61][62][63].
Another aspect of interest in the phylogenetic study of digeneans is the location of the maximum number of cortical microtubules in sperm. Based on the position of these microtubules, two groups of digeneans can be distinguished [26,37]: a group where the highest number of cortical microtubules is located in the anterior region of the spermatozoon and a group where the highest number of cortical microtubules is situated at an intermediate region of the sperm cell (respectively classified as type 1 and 2 according to Quilichini et al. [37]).
In P. characis, the maximum number of cortical microtubules is located in the anterior part of the spermatozoon, representing a type 1 spermatozoon according to Quilichini et al. [37]. This type is present in Allopodocotyle pedicellata and A. tunisiensis [28,29]. The remaining Opecoelidae species, except for Macvicaria obovata, exhibit a type 2 sperm cell, with the maximum number of cortical microtubules located in the middle part of the spermatozoon (see Table 1). In M. obovata, the maximum number of cortical microtubules is located in the nuclear region of the spermatozoon after the disappearance of the first axonemes [36].

Spine-Like Bodies
These triangular, electron-dense prominences containing a submembranous vesicle were first described in the opecoelid species O. furcatus by Miquel et al. [34]. The variability of their presence, absence, number, size and location in ornamented and/or non-ornamented areas has been widely discussed [26]. They are frequently observed in mature sperm cells of digenean species, including those in the Opecoelidae family (Table 1). However, spine-like bodies are absent from male gametes of H. fasciata [32] and P. characis (present study).

Mitochondrion
Mitochondria are a crucial component in digenean spermatozoa and are used to determine phylogenetic relationships. Their presence is considered a plesiomorphic character, while their absence is seen as a synapomorphy for the Eucestoda [18]. The formation of mitochondria in spermatozoa occurs during spermiogenesis, when numerous mitochondria accompany the nucleus' migration along the median cytoplasmic process [64]. The number, shape and location of mitochondria can be used to make phylogenetic inferences. The number of mitochondria in mature spermatozoa can range from one to three [26]. In the case of P. characis, male gametes have two mitochondria, in agreement with most studies on Opecoelidae spermatozoa. To date, the exception is the male gamete of O. furcatus and H. fasciata exhibiting only one mitochondrion [32,34].
The shape of mitochondria is a feature that varies among members of the Opecoelidae family. Different morphological types have been described, including tubular, moniliform and U-shaped posterior extremities. Bâ et al. [41] were the first to describe a moniliform mitochondrion in Holorchis micracanthum (Stossich, 1889), which appears as a series of mitochondrial bulges joined by a cord. This shape has also been reported in the spermatozoon of A. pedicellata and M. obovata [28,36]. Finally, a U-shaped posterior extremity of mitochondrion was described in A. tunisiensis by Kacem et al. [29].
The placement of mitochondria is considered an interesting aspect in most digeneans. In O. furcatus and H. fasciata [32,34], a single mitochondrion overlaps the anterior portion of the nucleus in the middle region of the cell. It always disappears before the posterior end of the second axoneme. In the remaining Opecoelidae species, two mitochondria are present: the first is located in the ornamented area of the spermatozoon and the second in the nuclear region. The level of their disappearance varies among the studied Opecoelidae species.
Peracreadium characis exhibits variability in the level of disappearance of the second mitochondrion, which can occur either before or after the posterior end of the second axoneme. This variability in the disappearance level of the second mitochondrion has also been described in A. tunisiensis [29], where it can occur both before and after the disorganization of the first axoneme. In L. gephyroberici and P. magnatestis [30,33], the second mitochondrion disappears after the posterior end of the second axoneme. In A. tunisiensis and H. epinepheli [29,31], it disappears at the same level as the second axoneme. In the other studied Opecoelidae, the second mitochondrion disappears before the posterior end of the second axoneme [23,28,[35][36][37][38].

Posterior Region of the Spermatozoon
The distal region of the sperm cell presents a highly variable morphology, and is considered to be a significant feature for establishing sperm models in digenean parasites.
In fact, Quilichini et al. [23] proposed three types of posterior spermatozoon extremities defined by the transition sequence of the second axoneme, cortical microtubules and nucleus. The Opecoelidean or type 1 sperm extremity presents the sequence 'axoneme, nucleus and cortical microtubules', whereas the Fasciolidean or type 2 sperm extremity presents the sequence 'cortical microtubules, axoneme and nucleus'. Finally, the Cryptogonimidean or type 3 extremity presents the sequence 'cortical microtubules, nucleus and axoneme'.
In P. characis, variability in the level of disappearance of the second mitochondrion can lead to variations in the sequence of sperm components toward the posterior extremity of the spermatozoon. This disappearance can occur either before or after the posterior end of the second axoneme. In fact, due to the non-conformity of posterior spermatozoon extremities of several species of digeneans to any of models proposed in Quilichini et al. [23], Bakhoum et al. [26] proposed other types of posterior spermatozoon extremities defined only by the last spermatozoon character instead of the sequence of characters in the extremities. Thus, according to Bakhoum et al. [26], the posterior extremity of the male gamete of P. characis belongs to type characterized by the presence of only cortical microtubules in the posterior extremity of the spermatozoon. This is a feature observed in the mature sperm of all opecoelids studied so far, except for P. magnatestis [30], which exhibits the nucleus.

Concluding Remarks: Contribution of Spermatological Characteristics to Opecoelidean Phylogenetic Inferences
Concerning the relationship between Opecoelidae and Brachycladioidea, differences in spermatozoa ultrastructural organization were reported between Opecoelidae and Acanthocolpidae (Stephanostomum murielae Bray & Justine, 2011 and Stephanostomoides tenuis (Manter, 1963) [69]), which are members of the Brachycladioidea. Therefore, the analysis of ultrastructural data of spermatozoa do not support a close relationship between the two groups.
Regarding the structure and organization of sperm cells in opecoelids, it has been found that they are quite similar. However, there are several discrepancies when analyzing the existing results in the different subfamilies. With regard to external ornamentation, all species follow pattern 2 as defined by Quilichini et al. [24], with the exception of P. characis (present study) and H. fasciata [32]. As for the posterior extremity, all studied species exhibit the opecoelidean type established by Quilichini et al. [23], with cortical microtubules as the terminal character, except for P. magnatestis [30], which is defined by the presence of the nucleus in the posterior tip of the male gamete. Despite the presence of anterior dense material in several opecoelid species, this character is absent in H. maculosus, N. testiobliqua, N. wisniewskii and O. furcatus [23,34,37,38].
Considering the digenean spermatozoa models proposed in Bakhoum et al. [26], model III describes the typical Opecoelidae sperm cell type. It is noteworthy that this pattern cannot be attributed to Allopodocotyle species (Hamacreadiinae) [28,29] because the largest number of cortical microtubules is located in an intermediate area of the sperm cell. Therefore, the studied Allopodocotyle species agree with the type IV model of digenean sperm cells. However, the other hamacreadiine studied to date (P. magnatestis) exhibits the type III spermatozoon [30]. In the subfamily Opistholebetinae, the studied species also show variability in the spermatozoon model. Thus, the sperm cell of M. obovata follows the type III whereas that of P. characis follows the type IV. The third studied opistholebetine, H. maculosus, has a doubtful or questionable location of the maximum number of cortical microtubules [23]. The remaining analyzed subfamilies, namely the Helicometrinae, Opecoelinae and Plagioporinae, are homogeneous in relation to the pattern of the sperm cell. The similarity in the ultrastructure of spermatozoa between P. characis (present study) and Allopodocotyle species [28,29] suggests a close relationship between the Opistholebetinae and Hamacreadiinae subfamilies, and this finding is supported by genetic studies conducted by Sokolov et al. [15]. However, using more extensive data, the phylogenetic analyses performed by Martin et al. [13] are more robust and do not show a close relationship between these subfamilies.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to internal laboratory policy.